Crystalline and amorphous forms of beta-l-deoxythymidine

ABSTRACT

Physical forms of beta-L-2′-deoxythymidine are disclosed that can be characterized by physical appearance, purity levels, Infra-Red and Raman spectroscopy, X-ray powder diffraction patterns, thermal properties, and methods of manufacture. These forms of beta-L-2′-deoxythymidine can be used in the manufacture of other forms of beta-L-2′-deoxythymidine, or in pharmaceutical compositions. Particularly preferred uses are in the treatment of hepatitis B.

CROSS-REFERENCE OF THE INVENTION

This application claims priority to U.S. Provisional Application Ser.No. 60/401,318, filed on Aug. 6, 2002.

FIELD OF THE INVENTION

The present invention describes crystalline and amorphous forms ofbeta-L-2′-deoxythymidine and mixtures thereof, substantially purecrystalline and is amorphous forms of beta-L-2′-deoxythymidine,analytical methods for confirming the presence and crystalline purity ofbeta-L-2′-deoxythymidine, pharmaceutical compositions thereof, and usesfor such compositions.

BACKGROUND OF THE INVENTION

The synthetic nucleoside beta-L-2′-deoxythymidine (L-dT) is known in theart. Antonin Holy first disclosed beta-L-2′-deoxythymidine and methodsfor preparing beta-L-2′-deoxythymidine in 1972 in “Nucleic AcidComponents and Their Analogs. CLIII. Preparation of2′-deoxy-L-Ribonucleosides of the Pyrimidine Series,” Collect. Czech.Chem. Commun. (1972), 37(12), 4072-87. The compound is represented bythe following chemical structure:

wherein R is H.

Several authors have reported antiviral activity ofbeta-L-2′-deoxythymidine against herpes simplex virus. See, e.g., Iottiet al., WO 92/08727; and Spadari et al., J. Med. Chem. (1992), 35(22),4214-20.

More recently, Gilles Gosselin, Jean-Louis Imbach and Martin Bryantdisclosed that the compound and its derivatives and analogues haveuseful properties against the hepatitis B virus. See WO 00/09531; PCTUS01/17301 and PCT US01/19147; U.S. Pat. Nos. 6,395,716; 6,569,837;6,444,652; and 6,566,344. L-dT is currently in advanced clinicaldevelopment by Idenix Pharmaceuticals, Inc. (Cambridge, Mass.).

Each of the foregoing references discloses a synthetic process forproducing L-dT that relies upon crystallizing the compound from ethanol.None of the references disclose the particular crystalline or physicalform of the L-dT that was obtained, or the water content of such L-dT.However, it has been recognized that crystalline, amorphous, hydrated,and various physical forms of the same compound can vary substantiallyin their biological properties, and in their ease of processing,manufacturing and/or pharmaceutical formulation. In addition, some formsare unstable under ambient conditions and thus require special storageand handling conditions to be maintained, or must be avoided altogether.

Given the commercial importance of beta-L-2′-deoxythymidine, it would beuseful to have additional forms and phases of beta-L-2′-deoxythymidinethat may exhibit beneficial properties in manufacturing or otherapplications. It is therefore an objective of this invention to providenovel forms and phases of beta-L-2′-deoxythymidine.

It is another objective to provide novel methods for the preparation andisolation of forms and phases of beta-L-2′-deoxythymidine.

It is still another objective of the invention to provide therapeuticuses of such forms and phases of beta-L-2′-deoxythymidine.

SUMMARY OF THE INVENTION

As a result of studies into the crystalline structure and variousphysical forms of L-dT, Applicants have unexpectedly made a number ofsignificant discoveries. In particular, Applicants have discovered:

-   -   structural forms of crystalline L-dT that can be characterized        by their physical appearance;    -   an amorphous form of L-dT; and    -   mixtures of these two forms.

These discoveries lead to the production of a form of L-dT that issubstantially devoid of water and can be maintained for a period oftime, or shelf life, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12months or more, by maintaining the compound under appropriate conditionsof low humidity. Given that nucleosides in general are polar, watersoluble compounds, and that L-dT in particular contains apyrimidinedione ring and two free hydroxy groups, it could not bepredicted that L-dT, when substantially devoid of waters of hydration,could be maintained for an extended shelf-life.

In addition, Applicants have discovered methods of analyzing the purityof crystalline L-dT for contamination by amorphous L-dT. Theseanalytical methods include diffractional, vibrational, thermal, andother analytical techniques. In still other embodiments the inventionincludes methods of making the foregoing forms of L-dT.

These forms of L-dT have advantageous properties in the preparation ofpharmaceutical compositions and in the treatment of hepatitis B.Beneficial properties can be attributed to these different physicalforms including increased solubility, improved bioavailability, ease ofchemical processing and/or ease of pharmaceutical formulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a XRPD (“X-ray powder diffraction”) pattern of crystallinebeta-L-2′-deoxythymidine and amorphous beta-L-2′-deoxythymidine.

FIG. 2 is a graphical depiction of the Infra-Red (IR) spectra ofcrystalline beta-L-2′-deoxythymidine and amorphousbeta-L-2′-deoxythymidine.

FIG. 3 is a graphical depiction of the Raman spectra of crystallinebeta-L-2′-deoxythymidine and amorphous beta-L-2′-deoxythymidine.

FIG. 4 is a graphical depiction of a TGA (“thermogravimetric analysis”)scan of crystalline beta-L-2′-deoxythymidine superimposed over a typicalDSC (“differential scanning calorimetry”) thermogram of the samecrystalline beta-L-2′-deoxythymidine, with an endotherm at 189.69° C.

FIG. 5 depicts in graphical form the moisture sorption and desorptiondata for crystalline beta-L-2′-deoxythymidine.

FIG. 6 is a graphical depiction of a TGA scan of amorphousbeta-L-2′-deoxythymidine superimposed over a typical DSC thermogram ofthe same amorphous beta-L-2′-deoxythymidine, with an endotherm at187.19° C.

FIG. 7 depicts a DSC thermogram obtained from temperature cyclingexperiments on amorphous beta-L-2′-deoxythymidine.

FIG. 8 depicts in graphical form the moisture sorption and desorptiondata for amorphous beta-L-2′-deoxythymidine.

DETAILED DESCRIPTION OF THE INVENTION Anhydrous L-dT Unassociated withWater

A principal embodiment of the present invention derives from thediscovery of crystalline beta-L-2′-deoxythymidine, and to methods ofpreventing the formation of hydrous forms that could affect thestability of the product. The inventors have surprisingly discoveredthat L-dT, crystallized from ethanol, and then maintained in anenvironment of less than or equal to about 45% relative humidity, and issubstantially impervious to water. The resulting crystalline L-dT isnon-solvated and free of adsorbed or absorbed water. As shown from theTG and DK curves in FIG. 4, when L-dT is crystallized and dried in anenvironment lacking humidity, it is devoid of waters of hydration andassociation (hence the lack of an endotherm until the substance melts atabout 189° C.). As FIG. 5 shows, however, crystalline L-dT willassociate with water (via adsorption, absorption or hydration) as therelative humidity exceeds about 45% to 55% relative humidity.

The fact that L-dT devoid of waters of hydration or association can evenbe obtained is surprising in view of the fact that L-dT is frequentlycrystallized from ethanol, which necessarily contains water as animpurity at levels greater than about 1, 2, or 3 vol. %, frequently upto about 5, 7, or 10 volume percent. These discoveries led to theinvention of L-dT which is substantially devoid of water for a period oftime, or shelf life, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11 or 12months or more, by maintaining the compound under appropriate conditionsof humidity. Because nucleosides in general are polar, water solublecompounds, and L-dT in particular contains a pyrimidinedione ring andtwo free hydroxy groups, two carbonyls, a secondary amine, a tertiaryamine, and an internal ether linkage, it could not be predicted thatL-dT that is devoid of waters of hydration or association could bemaintained for an extended shelf-life.

Therefore, in one embodiment the invention provides crystalline L-dTthat is substantially devoid of waters of hydration or association, forperiods of time exceeding 4, 6, 8, 10, or even 12 months. In anotherembodiment the invention provides a method of using crystalline L-dT issubstantially devoid of water at the end of the time period. The L-dT ispreferably administered to a human after such time period. Humidenvironments include environments in which the relative humidity exceedsgreater than about 10%, 20%, 30%, 40%, or 50%, but preferably not morethan about 75%, 65%, or 55% relative humidity. The periods of timeduring which the L-dT can be exposed to such humid environments aregreater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or even 12 months, butpreferably not more than 3 years, 2 years, or 1^(1/2) years. The term“humid environment” means the general ambient conditions to which L-dTor a container of L-dT is exposed. Relative humidities are taken as anaverage over the recited period of time.

This non-solvated crystalline form of beta-L-2′-deoxythymidine isprepared by crystallizing beta-L-2′-deoxythymidine from solution,optionally filtering said crystallized beta-L-2′-deoxythymidine, anddrying said beta-L-2′-deoxythymidine in an environment that lackshumidity (i.e. less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%relative humidity). The substance is preferably present in thesubstantial absence of amorphous L-dT.

The term hydrous, when used herein to describe a material, means thatthe weight of the material is increased by association with water,regardless of the mechanism of association. Thus, the term includeshydrated forms of the material, in addition to material in which wateris absorbed or adsorbed. Potential hydrated forms include themonohydrate, the dihydrate, the trihydrate, and the quaternary hydrate.

As used herein, the term, “substantially pure,” when used in referenceto a physical form, phase or crystalline form ofbeta-L-2′-deoxythymidine, refers to a phase or form ofbeta-L-2′-deoxythymidine which is greater than about 95% pure. Thismeans that the phase or form of beta-L-2′-deoxythymidine does notcontain more than about 5% of any other compound and, in one embodiment,does not contain more than about 5% of any other phase[s] or form[s] ofbeta-L-2′-deoxythymidine. In another embodiments, the term“substantially pure” includes a phase or form ofbeta-L-2′-deoxythymidine that is greater than about 96% pure. In stillanother embodiment the term “substantially pure” includes a phase orform of beta-L-2′-deoxythymidine that is greater than about 97%, 99%,99.5%, or 99.8% pure.

Similarly, the term “substantially in the absence of a second component”or “substantially free of a second component” when used in reference toa physical form, phase or crystalline form of beta-L-2′-deoxythymidine,refers to a phase or crystalline form of beta-L-2′-deoxythymidine thatdoes not contain more than about 5% of the second component. Morepreferably, the term “substantially in the absence of a secondcomponent” refers to a phase or form of beta-L-2′-deoxythymidine thatdoes not contain more than about 4% of the second component, and evenmore preferably no more than about 3%, 1%, 0.5%, or 0.2% of the secondcomponent.

Physical Forms of Crystalline L-dT Characterized by their PhysicalAppearance

In another principal embodiment the invention provides crystallinebeta-L-2′-deoxythymidine in various physical forms. Preferred physicalforms can be obtained using the methods more fully described in theExamples hereto and include:

white flakes;

clear aciculars;

white aciculars;

white solid;

white powder;

needles;

white chunks;

white fibers; and

white plates.

As used herein, the term “flake” is defined as a small flocculent massof crystals typically in the form of a thin flattened piece or layer.The term “acicular” means a material which incorporates within itselffine, needlelike crystals. A “powder” is a substance composed of fineparticles. “Needles” refer to slender pointed objects. A “chunk” refersto a short thick piece often crudely or roughly formed. A “fiber” refersto a structure or object resembling a thread. “Plates” refer to smooth,substantially flat and relatively thin pieces of material. A “solid”material refers to a uniform continuous mass of material obtained uponvaporization of solvent.

The differing physical properties of these physical forms can give riseto numerous unexpectedly beneficial properties including increasedsolubility, improved bioavailability, ease of chemical processing,and/or ease of pharmaceutical formulation.

In a preferred embodiment, one or more of the foregoing forms is presentin its preferred orientation. Preferred orientation is the tendency forcrystals, usually plates or needles, to align themselves with somedegree of order. In various embodiments, the foregoing physical formsare further characterized by purity levels. For example, in oneembodiment the crystalline beta-L-2′-deoxythymidine is substantiallyfree of amorphous beta-L-2′-deoxymymidine.

Still other embodiments relate to methods of manufacturing the foregoingphysical forms of beta-L-2′-deoxythymidine. These methods generallyrequire dissolution of beta-L-2′-deoxythymidine in an appropriatesolvent, followed by crystallization using one of various techniques. Aparticularly preferred solvent is ethanol, and especially absoluteethanol, although other solvents can be employed including acetone,acetonitrile, dichloromethane, N,N-dimethylformamide, ethyl acetate,ethyl ether, 1,1,1,3,3,3-hexafluoroisopropanol, hexane, isoproponal,methanol, tetrahydrofuran, toluene, 2,2,2-trifluoroethanol, and water,or mixtures of the foregoing solvents with water.

While numerous methods for crystallizing a solute are known and can beemployed in the practice of this invention, particularly preferredmethods of recrystallization include:

-   -   crystallizing said dissolved beta-L-2′-deoxythymidine via fast        evaporation (i.e. evaporation in an open container under ambient        conditions);    -   crystallizing said dissolved beta-L-2′-deoxythymidine via slow        evaporation (i.e. evaporation in a substantially closed        container containing only small holes for escape of vapors under        ambient conditions);    -   crystallizing said dissolved beta-L-2′-deoxythymidine via slow        cooling from an elevated temperature (preferably of about 45-60°        C.) under substantially ambient conditions (the dissolution is        preferably performed at an elevated temperature, followed by        rapid filtration of the dissolved beta-L-2′-deoxythymidine        before the slow cooling);    -   crystallizing said dissolved beta-L-2′-deoxythymidine via crash        cooling;    -   crystallizing said dissolved beta-L-2′-deoxythymidine via slurry        methods.

Ambient conditions in any of the methods of crystallization can bealtered in terms of temperature (i.e. greater than 10° C., 20° C., or30° C. than room temperature), or humidity (i.e. less than 45%, 35%, or25% relative humidity).

Amorphous L-dT

An amorphous and hygroscopic form of beta-L-2′-deoxythymidine is alsodisclosed that can be distinguished from crystallinebeta-L-2′-deoxythymidine by Infra-Red and Raman spectroscopy, X-raypowder diffraction patterns, thermal properties, and methods ofmanufacture. The amorphous phase of beta-L-2′-deoxythymidine exhibitssignificant hygroscopicity and improved solubility when compared tocrystalline beta-L-2′-deoxythymidine, a fact which could result inenhanced bioavailability in vivo. This form of beta-L-2′-deoxythymidinecan be used in the manufacture of other forms ofbeta-L-2′-deoxythymidine, or in pharmaceutical compositions.Particularly preferred uses of this form are the treatment of hepatitisB.

The amorphous and hygroscopic form of beta-L-2′-deoxythymidine ischaracterized by the IR and Raman spectra in FIGS. 2 and 3, the TGA andDSC curves in FIG. 6, the DSC temperature cycling characteristics shownin FIG. 7, the moisture sorption data shown in FIG. 8, and/or the X-raypowder diffraction pattern in FIG. 1. This form should be contrastedwith crystalline beta-L-2′-deoxythymidine, which can be characterized bythe IR and Raman spectra in FIGS. 2 and 3, the TGA and DSC curves inFIG. 4, the moisture sorption data shown in FIG. 5, and/or the X-raypowder diffraction pattern shown in FIG. 1. Amorphous and crystallineforms of beta-L-2′-deoxythymidine are further characterized in theExamples contained herein.

Thus, the invention further provides:

-   -   a) an amorphous and hygroscopic phase of        beta-L-2′-deoxythymidine, as characterized by X-ray powder        diffraction analysis, physical properties, and methods of        manufacture;    -   b) processes for making an amorphous and hygroscopic phase of        beta-L-2′-deoxythymidine;    -   c) therapeutic and/or pharmaceutical compositions of the        amorphous and hygroscopic phase of beta-L-2′-deoxythymidine,        optionally in the presence of a pharmaceutical acceptable        carrier, and    -   d) novel therapeutic uses for the amorphous and hygroscopic        phase of beta-L-2′-deoxythymidine, especially in the treatment        of viral diseases such as hepatitis B.

Amorphous beta-L-2′-deoxythymidine is obtained when meltedbeta-L-2′-deoxythymidine is rapidly quenched to below about 40 or 50° C.A DSC temperature cycling thermogram of amorphousbeta-L-2′-deoxythymidine is presented in FIG. 7, which shows that theglass transition temperature for this phase is about 50° C.

Low temperatures and low levels of humidity are preferably observed whenstoring amorphous beta-L-2′-deoxythymidine. For example, it is preferredto store amorphous beta-L-2′-deoxythymidine at temperatures below 50,40, 30, 20, 10, or 0° C. Moreover, it is preferred to store theamorphous beta-L-2′-deoxythymidine at relative humidities of less than25%, 20%, 15%, 10%, or 5%.

In another embodiment, the invention provides amorphousbeta-L-2′-deoxythymidine in substantially pure form i.e., in the absenceof crystalline beta-L-2′-deoxythymidine. In still another embodiment theinvention provides a pharmaceutical composition that comprises amorphousbeta-L-2′-deoxythymidine, further comprising a pharmaceuticallyacceptable carrier.

Analytical Methods of Assuring the Purity of Crystalline L-dT

In view of the stability issues surrounding amorphousbeta-L-2′-deoxythymidine, it is very important to ensure during themanufacturing process that batches of crystalline L-dT are notinadvertently contaminated by the amorphous form. Thus, otherembodiments of the present invention relate to methods of detectingcrystalline beta-L-2′-deoxythymidine contaminated by the amorphous formof beta-L-2′-deoxythymidine, and to methods of manufacturing crystallinebeta-L-2′-deoxythymidine that is substantially free of amorphousbeta-L-2′-deoxythymidine. As can be seen by a comparison of FIGS. 2 and3, crystalline and amorphous beta-L-2′-deoxythymidine can each becharacterized by IR and Raman spectrometry, and the two forms can bedistinguished by peak broadening that occurs from the crystalline to theamorphous form. These peak broadenings are observed between 2200-2600;1600-1800, 1000-1500, or 500-1000 cm⁻¹ in an IR spectra, and between2800-3200, 1600-1675, 1300-1500, or 700-900 in a Raman spectra.

Therefore, in various embodiments the invention provides a method ofdetecting amorphous contamination of beta-L-2′-deoxythymidine, ordistinguishing between crystalline and amorphousbeta-L-2′-deoxythymidine, comprising:

-   -   subjecting a material that contains beta-L-2′-deoxythymidine to        IR spectrometry; and    -   evaluating breadth of peaks displayed between 2200-2600,        1600-1800, 1000-1500, or 500-1000 cm⁻¹ by reference to a known        IR spectra for crystalline beta-L-2′-deoxythymidine.

In other embodiments the invention provides a method of detectingamorphous contamination of beta-L-2′-deoxythymidine, or distinguishingbetween crystalline and amorphous beta-L-2′-deoxythymidine, comprising:

-   -   subjecting a material that contains beta-L-2′-deoxythymidine to        Raman spectrometry; and    -   evaluating the breadth of peaks displayed between 2800-3200,        1600-1675, 1300-1500, or 700-900 cm⁻¹ by reference to a known        Raman spectra for crystalline beta-L-2′-deoxythymidine.

In still other embodiments the invention relates to the manufacture ofpharmaceutical compositions of crystalline beta-L-2′-deoxythymidinesubstantially in the absence of the amorphous form. Thus, the inventionprovides a method of manufacturing pharmaceutical formulations ofcrystalline beta-L-2′-deoxythymidine comprising:

-   -   crystallizing two or more batches of beta-L-2′-deoxythymidine        from solution;    -   subjecting the batches to IR spectrometry;    -   selecting for further processing the batches that do not display        significant breadth of peaks (i.e. breadth consistent with the        presence of amorphous L-dT) between 2200-2600, 1600-1800,        1000-1500, or 500-1000 cm″¹ from a known IR spectra for        crystalline beta-L-2′-deoxythymidine; and    -   formulating said selected batches into pharmaceutical        formulations of beta-L-2′-deoxythymidine.

In another embodiment, the invention provides a method of manufacturingpharmaceutical formulations of crystalline beta-L-2′-deoxythymidinecomprising:

-   -   crystallizing two or more batches of beta-L-2′-deoxythymidine        from solution;    -   subjecting the batches to Raman spectrometry;    -   selecting for further processing the batches do not display        significant breadth of peaks (i.e. breadth consistent with the        presence of amorphous L-dT) between 2800-3200, 1600-1675,        1300-1500, or 700-900 cm⁻¹ from a known Raman spectra of        crystalline beta-L-2′-deoxythymidine; and    -   formulating said selected batches into pharmaceutical        formulations of beta-L-2′-deoxythymidine.

EXAMPLES Example 1 Stereoselective Chemical synthesis ofbeta-L-2′-deoxythymidinc

A mixture of compound 5 (105.8 g, 0.242 mol), iodine (76.8 g), CAN (66.4g) and acetonitrile (2550 ml) was stirred at 80° C. for 3 h then thereaction mixture was cooled at room temperature leading tocrystallization of compound 7 (86.6 g, 63.5%); m. p. 192-194° C.; ¹H NMR(DMSO) δ ppm. 8.34 (s, IH, NH), 8.2-7.2 (m, 11H, 2 C₆H₅CO, H-6), 6.31(q, 1H, H-1 ′, J=5.5 Hz and J=8.7 Hz), 5.5 (m, IH, H-3′), 4.7 (m, 2H,H-5′, H-5″), 4.5 (m, 1H, H-4′), 2.7 (m, IH, H-2′), 2.3 (m, IH, H-2″);FAB<0, (GT) m/e 561 (M−H)⁻, 237 (B)⁻; FAB>0 (GT) 563 (M+H)⁺; [α]_(D)²⁰+39.05 (c=1.05 in DMSO); UV (EtOH 95)υ_(max)=281 nm(ε=9000)υ_(min)=254 nm (ε=4000)υ_(max)=229 nm (ε=31000); Anal. Calcd forC₂₃H₁₉IN₂O₇: C, 49.13; H, 3.41; N, 4.98; I, 22.57. Found: C, 49.31; H,3.53; N, 5.05; I, 22.36.

Reagents; Iodine: Fluka, 99.8%, ref 57650

Cerium ammonium nitrate (CAN): Aldrich, >98.5%, ref 21,547-3

3′5′-Di-O-benzoyl-2′-deoxy-3-N-toluoyl-β-L-thymidine (2)

To a solution of compound 7 (86.6 g, 0.154 mol) in anhydrous pyridine(1530 ml) containing N-ethyldiisopropylamine (53.6 ml) was added,portionwise at 0° C., p-toluoyl chloride (40.6 ml). The reaction mixturewas stirred for 2 h at room temperature, then water was added to stopthe reaction and the reaction mixture was extracted with methylenechloride. The organic phase was washed with water, dried over sodiumsulfate and evaporated to dryness to give crude3′,5′-di-O-benzoyl-2′-deoxy-3-N-toluoyl-5-iodo-β-L-uridine (8 which canbe used for the next step without further purification.

A solution of the crude mixture 8, palladium acetate (3.44 g),triphenylphosphine (8.0 g) in N-methylpyrrolidinone (1375 ml) withtriethylamine (4.3 ml) was stirred at room temperature for 45 nm. Then,tetramethyltin (42.4 ml) was added dropwise at 0° C. under argon. Afterstirring at 100-110° C. overnight, the reaction mixture was poured intowater and extracted with diethyl ether. The organic solution was driedover sodium sulfate and concentrated under reduced pressure. The residuewas purified by a silica gel column chromatography [eluant: stepwisegradient of ethyl acetate (0-10%) in toluene] to give compound 9 as afoam (42.3 g, 48.3% for the 2 steps). ¹H NMR (DMSO) δ ppm. 8.3-7.2 (m,15H, 2 C₆H₅CO, 1 CH₃C₆H₄CO, H-6), 6.29 (t, 1H, H-1 ′, J=7.0 Hz), 5.7 (m,1H, H-3′), 4.7-4.5 (m, 3H, H-5′, H-5″, H-4′), 2.7-2.6 (m, 2H, H-2′,H-2″); FAB<0, (GT) m/e 567 (M−H)⁻, 449 (M-CH₃C₆H₄CO)⁻, 243 (B)⁻121(C₆H₅COO); FAB>0 (GT) 1137 (2M+H)⁺, 569 (M+H)⁺, 325 (M−B)⁻245 (B+2H)⁺,119 (CH₃C₆H₅CO)⁺.

Reagents:

p-Toluoyl chloride, Aldrich, 98%, ref 10, 663-1

Diisopropylethylamine: Aldrich, >99.5%, ref 38, 764-9

N-methylpyrrolidinone: Aldrich, >99%, ref 44, 377-8

Palladium acetate: Aldrich, >99.98%, ref 37, 987-5

Triphenylphosphine: Fluka, >97%, ref 93092

Tetramethyltin: Aldrich, >99%, ref 14,647-1

2′-Deoxy-p-L-thymidine

A solution of compound 9 (42.3 g, 0.074 mol) in methanol saturated withammonia (1850 ml) was stirred at room temperature for two days. Afterevaporation of the solvent, the residue was diluted with water andwashed several times with ethyl acetate. The aqueous layer wasseparated, evaporated under reduced pressure and the residue waspurified by a silica gel column chromatography [eluant: stepwisegradient of methanol (0-10%) in methylene chloride] to give pure2′-deoxy-β-L-thymidine (11.62 g, 64.8%) which was crystallized fromethanol; m.p. 185-188° C.; ¹H NMR (DMSO) δ ppm 11.3 (s, IH, NH), 7.70(s, IH, H-6), 6.2 (pt, IH, H-1′), 5.24 (d, IH, OH-3′, J=4.2 Hz), 5.08(t, IH, OH-5′, J=5.1 Hz), 4.2 (m, IH, H-3′), 3.7 (m, IH, H-4′), 3.5-3.6(m, 2H, H-5′, H-5″), 2.1-2.0 (m, 2H, H-2′, H-2″); FAB<0, (GT) m/e 483(2M−H)⁻, 349 (M+T−H)⁻241 (M−H)⁻125 (B)⁻; FAB>0 (GT) 243 (M+H)⁺, 127(B+2H)⁺;)⁺; [α]D²⁰-13.0 (c-1.0 in DMSO); UV (pH 1) υ_(max)=267 nm(ε=9700), υ_(min)=234 nm (υ=2000).

Reagent:

Methanolic ammonia: previously saturated at −5° C., tightly stoppered,and kept in a freezer.

Example 2 Characterization of Physical Forms of L-dT ExperimentalMethods Crystallization Studies

A weighed sample of L-dT (usually 20-30 mg) was treated with aliquots ofthe test solvent. Solvents were either reagent or HPLC grade. Thealiquots were usually 100 μL or 500 μL. Between additions the mixturewas sonicated. When the solids dissolved, as judged by visualinspection, the solution was filtered, then left in an open vial underambient conditions (fast evaporation). The procedure was repeated, andthe resulting solution was left under ambient conditions in a vial withaluminum foil containing pinholes (slow evaporation). Samples were alsogenerated by filtering a warm solution of L-dT into a cold antisolvent(cold precipitation).

Solubilities were estimated from these experiments based on the totalsolvent used to give a solution. Note that actual solubilities may begreater than those calculated because of the use of too-large solventaliquots or a slow rate of dissolution. If the dissolution did not occurduring the experiment the solubility is expressed as “less than.” If thesolid dissolved before the whole aliquot of solvent was added thesolubility is listed as “greater than.”

The solid was also dissolved in a test solvent at an elevatedtemperature (either 45 or 60° C.). The resulting solution was rapidlyfiltered into a vial kept on the same hotplate. The heat source was thenturned off and the hotplate and vial were allowed to cool to roomtemperature (slow cool). The vial was then allowed to stand at ambienttemperature overnight. The presence or absence of undissolved solids wasnoted; if there were no solids present, or an amount of solid judged toosmall for XRPD analysis, the vial was placed in a refrigeratorovernight. Again the presence or absence of undissolved solids was notedand, if there were none, the vial was placed in a freezer overnight.Solids were removed by filtration and allowed to dry in the air.

Slurry experiments were carried out by making saturated solutionscontaining excess solid. The slurries were agitated at ambienttemperature. The insoluble solids were recovered by filtration.Crystallizations using antisolvent addition were also performed. Solidwas dissolved in a solvent and filtered into an antisolvent at either 0°C. or −78° C. If no solids immediately formed, the samples were eitherleft to stand under ambient conditions and/or the solvent was removed byrotary evaporation. Any solids formed were removed by filtration andair-dried. Vapor diffusion experiments involved placing a saturatedsolution of L-dT in a vial. The vial was placed in a larger vialcontaining an antisolvent which was then sealed and kept at ambienttemperature. Samples were also generated by cooling of a melt of L-dT toroom temperature and also lyophilization using water.

All samples generated as described above were air-dried and analyzed byX-ray powder diffraction (XRPD).

X-ray Powder Diffraction

X-ray powder diffraction (XRPD) analyses were performed using a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation. Theinstrument is equipped with a fine focus X-ray tube. The tube voltageand amperage were set to 40 kV and 40 mA, respectively. The divergenceand scattering slits were set at 1° and the receiving slit was set at0.15 mm. Diffracted radiation was detected by a NaI scintillationdetector. A theta-two theta continuous scan at 3°/min (0.4 sec/0.02°step) from 2.5 to 40° 20 was used. A silicon standard was analyzed tocheck the instrument alignment. Data were collected and analyzed usingXRD-6000 v. 4.1. Samples were prepared for analysis by placing them in asilicon sample holder or aluminum holder with silicon insert.

Characteristic powder X-ray diffraction pattern peak positions arereported for crystalline forms in terms of the angular positions (twotheta) within an allowable variability of plus or minus 0.1°. Thisallowable variability is specified by the US Pharmacopeia, pages1843-1844 (1995). The variability of plus or minus 0.1° is intended tobe used when comparing two powder X-ray diffraction patterns. Inpractice, if a diffraction pattern peak from one pattern is assigned arange of angular positions (two theta) which is a measured peak positionplus or minus 0.1° and a diffraction pattern peak from the other patternis assigned a range of angular positions (two theta) which is themeasured peak position plus or minus 0.1° and if those ranges of peakpositions overlap, then the two peaks are considered to have the sameangular position (two theta). For example, if a diffraction pattern peakfrom one pattern is determined to have a peak position of 5.20°, forcomparison purposes the allowable variability allows the peak to beassigned a position in the range of 5.10°-5.30°. If a comparison peakfrom the other diffraction pattern is determined to have a peak positionof 5.35°, for comparison purposes the allowable variability allows thepeak to be assigned a position in the range of 5.25°-5.45°. Becausethere is overlap between the two ranges of peak positions, the two peaksbeing compared are considered to have the same angular position (twotheta).

Thermal Analysis

Thermogravimetric (TG) analyses were performed using a TA Instruments2050 or 2950 thermogravimetric analyzer. Each sample was placed in analuminum sample pan and inserted into the TG furnace. Samples were firstequilibrated at 25° C., then heated under nitrogen at a rate of 10°C./min, up to a final temperature of 350° C. Nickel and Alumel™ wereused as the calibration standards.

Differential scanning calorimetry (DSC) was performed using a TAInstruments differential scanning calorimeter 2920. The sample wasplaced into an aluminum DSC pan, and the weight accurately recorded. Thepan was covered with a lid and left uncrimped. Each sample wasequilibrated at 25° C. and heated under a nitrogen purge at a rate of10° C./min, up to a final temperature of 350° C. For studies of theglass transition temperature (T_(g)) of the amorphous material, thesample was heated at a rate of 20° C./min, up to 90° C. and held therefor 3 minutes. The sample was then allowed to cool and equilibrate at 0°C. The sample was again heated at a rate of 20° C./min, up to 90° C.,held there for 3 minutes, and then cooled to 0° C. The sample was thenheated at 20° C./min up to a final temperature of 225° C. Indium metalwas used as the calibration standard.

Hot stage microscopy was performed using a Linkam hot stage (model FTIR600) mounted on a Leica DMLP microscope equipped with a Sony DXC-970MD3CCD camera for collecting images. A 10× objective was used with crosspolarizers to view samples. Samples were placed on a glass coverslip. Acoverslip was then placed over the sample. Each sample was visuallyobserved as the stage was heated. Images were captured using Linksysversion 2.27 (Linkam). The hot stage was calibrated using USP meltingpoint standards.

Spectroscopy

Infrared spectra were acquired on a Magna-IR 560®/or 860® Fouriertransform infrared (FT-IR) spectrophotometer (Thermo Nicolet) equippedwith an Ever-Glo mid/far IR source, potassium bromide (KBr)beamsplitter, and a deuterated triglycine sulfate (DTGS) detector. Adiffuse reflectance accessory (the Collector™, Thermo Spectra-Tech) wasused for sampling. Each spectrum represents 256 co-added scans collectedat a spectral resolution of 4 cm⁻¹. Sample preparation consisted ofplacing the sample into a 3-mm diameter cup and leveling the materialwith a frosted glass slide. A background data set was acquired with analignment mirror in place. A Log 1/R(R=reflectance) spectrum wasacquired by taking a ratio of these two data sets against each other.Wavelength calibration was performed using polystyrene.

FT-Raman spectra were acquired on an FT-Raman 960 spectrometer (ThermoNicolet). This spectrometer uses an excitation % wavelength of 1064 nm.Approximately 0.5 W of Nd:YVO₄ laser power was used to irradiate thesample. The Raman spectra were measured with an indium gallium arsenide(InGaAs) detector. The samples were prepared for analysis by placing thematerial in a glass tube and positioning the tube in a gold-coated tubeholder in the accessory. A total of 256 sample scans were collected from100-3600 cm⁻¹ at a spectral resolution of 4 cm⁻¹, using Happ-Genzelapodization. Wavelength calibration was performed using sulfur andcyclohexane.

Moisture Sorption/Desorption

Moisture sorption/desorption data were collected on a VTI SGA-I00 VaporSorption Analyzer. Sorption and desorption data were collected over arange of 5% to 95% relative humidity (RH) at 10% RH intervals under anitrogen purge. Samples were not dried prior to analysis. Equilibriumcriteria used for analysis were less than 0.0100% weight change in 5minutes, with a maximum equilibration time of 3 hours if the weightcriterion was not met. Data were not corrected for the initial moisturecontent of the samples. NaCl and PVP were used as calibration standards.

Results Approximate Solubilities

Approximate solubilities of L-dT in various solvents at ambienttemperature were determined during the crystalline screen, and aresummarized in Table 1. L-dT was found to be most soluble in 1:1THF:water (127 mg/mL), followed by N,N-dimethylformamide (72 mg/mL), 1:1methanol:water (58 mg/mL), 4:1 acetone:water (51 mg/mL), water (32mg/mL), 1,1,1,3,3,3-hexafluoro-2-propanol (22 mg/mL), methanol (11mg/mL), 2,2,2-trifluoroethanol (8 mg/mL) and EtOH (>4 mg/mL). L-dT issparingly soluble in acetone, acetonitrile, ethyl acetate, diethylether, isopropanol, methylene chloride, and toluene

Crystallization Studies

Crystallization studies were also carried out to generate differentsolid forms of L-dT. The technique involved generation of solid L-dTunder a variety of conditions and characterization of the resultingsamples by XRPD. The experiments performed are summarized in Tables 2and 3. One distinct crystalline XRPD pattern was found, termedcrystalline L-dT, as well as an amorphous form. Amorphous material wasgenerated by quenching a melt of the material on a cold countertop orlyophilization from aqueous solution. Many of the generated samplesexhibited preferred orientation. Preferred orientation can affect peakintensities, but not peak positions, in XRPD patterns. The sectionsbelow summarize the characterization of the crystalline and amorphousmaterials.

Characterization of Crystal Form

The XRPD pattern of the initial material received for thecrystallization screen is shown in FIG. 1, and was termed representativeof crystalline L-dT. Almost all of the crystallizations performed duringthe course of the screen resulted in crystalline L-dT material. The IRand Raman spectra for crystalline L-dT are shown in FIGS. 2 and 3,respectively.

The thermal data for crystalline L-dT is shown in FIG. 4 and listed inTable 4. The TG curve displays minimal weight loss up to 175° C.,indicating an unsolvated material. The DSC curve displays an endothermat 190° C. The moisture sorption data for crystalline L-dT is shown inFIG. 5 and summarized in Table 5. The material gains a relatively smallamount of weight up to 45% RH, but above this point the material gainsweight up to a total of 11.1% at 95% RH. All of this weight is lost uponlowering the relative humidity to 5% RH.

Characterization of Amorphous Form

Amorphous material was prepared by quenching a melt of the material toroom temperature or by lyophilization from aqueous solutions. The IR andRaman spectra of amorphous are shown in FIG. 2 and FIG. 3, respectively.The IR spectra for the amorphous material differs from the crystallinematerial in that the peaks from the amorphous material are broader andvary in shape between 1500-1900 cm⁻¹. The peaks in the Raman spectrum ofthe amorphous material also are broader than those seen for thecrystalline material; however, the amorphous Raman spectrum displayssignificant peak shifts around 2800-3200, 1600-1675, 1300-1500, and700-900 cm⁻¹.

The thermal data for the amorphous is shown in FIG. 6. The TG curveshows a 0.5% weight loss up to 175° C. The material displays anexothermic event at about 135° C., which is likely a crystallization,followed by a melting endotherm at about 187° C. A DSC temperaturecycling experiment shown in FIG. 7, demonstrates that the glasstransition temperature for the amorphous material is ˜50° C.

The moisture sorption data for amorphous L-dT is shown in FIG. 8. Thematerial loses ˜0.4% of its weight upon equilibration at about 5% RH,and gains ˜2.3% up to about 55% RH. Above this point, the material losessome of this weight and continues to gain weight above about 85% RH. Thematerial loses almost all of this weight upon lowering the relativehumidity from about 95% RH 10 to about 5% RH. The XRPD pattern of thematerial after the experiment matches that of crystalline L-dT. It islikely that the sudden weight drop above about 55% RH is indicative of arecrystallization and the concomittant loss of water.

TABLE 1 Approximate Solubilities of crystalline L-dT Solvent Solubility(mg/mL)′ acetone <4 acetone:water (4:1) 51 acetonitrile (ACN) <4dichloromethane <3 N,N-dimethylformamide (DMF) 72 ethanol (EtOH) >3.6*ethyl acetate (EtOAc) <4 ethyl ether <31,1,1,3,3,3-hexafluoroisopropanol 22 hexane <4 isopropanol (IPA) <3methanol (MeOH) 11 MeOH:water (1:1) 58 tetrahydrofuran (THF) 4THF:water(1:1) 127 toluene <4 2,2,2-trifluoroethanol 8 water 32^(a)Solubilities are calculated based on the total solvent used to givea solution; actual solubilities may be greater because of the volume ofthe solvent portions utilized or a slow rate of dissolution. If a clearsolution was not obtained the values for the solubilities are listed as“less than.” If the solid dissolved before the whole aliquot of solventwas added the solubility is listed as “greater than.” Values are roundedto the nearest mg/mL. *after 2 hours, all solid was dissolved.

TABLE 2 Crystallization Screen of L-dT Solvent Conditions^(a) Habit XRPDResults^(b) acetone slurry small white aciculars crystalline acetone 1day slurry needles crystalline (50° C.) acetone:water(4:1) FE unknowncrystalline 1 day slurry needles crystalline (50° C.) acetonitrileslurry small white aciculars crystalline 1 day slurry needlescrystalline (50° C.) dichloromethane slurry small white acicularscrystalline diethyl ether slurry small white aciculars crystallineN,N-dimethyl-formamide FE white chunks crystalline (DMF) SE whiteneedles, crystalline(PO) chunks SC (60° C.) no solid — ethanol FE whiteflakes, chunks crystalline SE clear aciculars crystalline SC (60° C.)unknown white crystalline solid CC white solid crystalline 1 day slurryneedles crystalline (50° C.) ethyl acetate slurry small white platescrystalline Hexaflurorisopropanol FE small white flakes crystalline SEwhite flakes crystalline SC (60° C.) no solid — rotovap white solidcrystalline hexanes slurry small white aciculars crystalline isopropanolslurry white powder crystalline 1 day slurry needles crystalline (50°C.) methanol FE white needles crystalline SE white needles, flakescrystalline SC (60° C.) white chunks crystalline CC white solidcrystalline rotovap white solid crystalline MeOH:water(1:1) FE needlescrystalline 1 day slurry no solids — (50° C.) no solids — CC freezertoluene slurry white powder crystalline tetrahydrofuran FE clumps ofwhite crystalline fibers (THF) SE white aciculars crystalline(PO) SC(60° C.) white needles crystalline CC white solid crystalline rotovapwhite solid crystalline THF:Water(1:1) FE needles crystalline 1 dayslurry no solids — (50° C.) CC no solids — freezer2,2,2-trifluoroethanol FE white needles, flakes crystalline SE whiteneedles crystalline SC (60° C.) no solid — rotovap white solidcrystalline water FE unknown white solid crystalline SE clear acicularscrystalline SC (60° C.) no solid — 1 day slurry needles crystalline (50°C.) freeze dry white solid crystalline freeze dry fluffy white solidamorphous freeze dry — crystalline melt/quench clear solid amorphousmelt/quench clear solid crystalline + amorphous melt/quench clear solidamorphous melt/quench clear solid amorphous melt/quench clear solidamorphous ^(a)FE = fast evaporation; SE = slow evaporation; SC = slowcool, CC = crash cool, rotovap = rotary evaporator ^(b)PO = preferredorientation; LC = low crystallinity; SS = small sample; IS =insufficient sample

TABLE 3 Cold Precipitation Crystallizations Solvent Anti-solvent HabitXRPD Results N,N- acetone no solid — dimethylformamide toluene whitesolid crystalline ethanol acetone no solid — ethyl acetate white solidcrystalline methanol acetone no solid — dichloromethane white solidcrystalline diethyl ether white solid crystalline tetrahydrofuranacetone no solid — diethyl ether white solid crystalline hexanes whitesolid crystalline water acetone no solid — acetonitrile no solid —isopropanol no solid — *solid dissolved in solvent at 60° C., hotfiltered into vial containing cold antisolvent (cooled indry-ice/acetone slurry) None of these yielded solid immediately, solidformed after samples had been in freezer several days.

TABLE 4 Thermal Data for L-dT Forms Form DSC Results (° C.)^(a) TGResults^(b) crystalline endo 190 <0.1 amorphous transition ~44, exo 135,0.5 endo 187 amorphous T_(g) ~50 — ^(a)maximum temperature reported;endo = endotherm; exo = exotherm; T_(g) = glass transition ^(b)percentvolatiles measured at 175° C.

TABLE 5 Summary of Moisture Sorption/Desorption Data for For CrystallineL-dT Solid Forms Lot No. Moisture Balance Results XRFD Results 1 <0.1%weight loss at 5% RH crystalline 1.1% weight gain at 95% RH 1.1% lossfrom 95-5% RH 2 −0.4% weight loss at 5% RH crystalline 2.8% weight gainat 95% RH 2.7% loss from 95-5% RH

Pharmaceutical Compositions

Humans suffering from viral diseases such as HBV can be treated byadministering to the patient an effective amount of the forms of thecompound of the present invention, or a pharmaceutically acceptableprodrug or salt thereof in the presence of a pharmaceutically acceptablecarrier or diluent. The active materials can be administered by anyappropriate route, for example, orally, parenterally, or intravenously,in liquid or solid form.

A preferred dose of the compound for HBV will be in the range from about1 to 75 mg/kg, preferably 1 to 50 or 20 mg/kg, of body weight per day,more generally 0.1 to about 100 mg per kilogram body weight of therecipient per day. The effective dosage range of the pharmaceuticallyacceptable salts and prodrugs can be calculated based on the weight ofthe parent nucleoside to be delivered. If the salt or prodrug exhibitsactivity in itself, the effective dosage can be estimated as above usingthe weight of the salt or prodrug, or by other means known to thoseskilled in the art.

The compound is conveniently administered in any suitable dosage form,including but not limited to one containing 7 to 3000 mg, preferably 70to 1400 mg of active ingredient per unit dosage form. An oral dosage of50-1000 mg is usually convenient.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrug or salts thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, antifungals, anti-inflammatories, or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A non-solvated crystalline form of beta-L-2′-deoxythymidine devoid ofwaters of association, having less than 0.1% weight loss at 5% relativehumidity, 1.1% weight gain at 95% relative humidity and 1.1% weight lossfrom 95%-5% relative humidity.
 2. A non-solvated crystalline form ofbeta-L-2′-deoxythymidine devoid of waters of association and having theX-ray powder diffraction pattern shown in FIG.
 1. 3. Thebeta-L-2′-deoxythymidine of claim 1 or 2 prepared by crystallizingbeta-L-2′-deoxythymidine from solution, optionally filtering saidcrystallized belta-L-2′-deoxythymidine, and drying saidbeta-L-2′-deoxythymidine in an environment comprising less than about40% relative humidity.
 4. A pharmaceutical formulation comprising thenon-solvated crystalline form of beta-L-2′-deoxythymidine according toclaim 1 or
 2. 5. The pharmaceutical formulation according to claim 4 inan oral solid form.
 6. The oral solid formulation of claim 5 furthercomprising at least one of an inert diluent, an edible carrier,excipient, a compatible binding agent, glidant, lubricant or adjuvantmaterial.
 7. The oral solid formulation of claim 6 wherein the bindingagent is microcrystalline cellulose, the excipient is starch, theglidant is colloidal silicon dioxide, and the lubricant is magnesiumstearale.
 8. The oral solid formulation of claim 6 in the form of atablet, troche or capsule.
 9. The oral solid formulation of claim 8having a dosage unit of 50-1000 mg.
 10. The oral solid formulation ofclaim 8 in the form of a tablet having a dosage unit of 50-1000 mg. 11.The tablet of claim 10 comprising a coating of sugar, shellac or otherenteric agent.
 12. A method of treating hepatitis B in a human subjectcomprising administering the oral solid formulation of claim
 9. 13. Amethod of treating hepatitis B in a human subject comprisingadministering the oral solid formulation of claim
 10. 14. A method oftreating hepatitis B virus comprising administering to a human afflictedwith Hepatitis B virus the oral solid formulation of claim
 9. 15. Amethod of treating hepatitis B virus comprising administering to a humanafflicted with Hepatitis B virus the oral solid formulation of claim 10.